Immunity to folate receptors

ABSTRACT

This document provides methods and materials related to assessing immunity to folate receptors. For example, methods and materials for assessing FRα immunity in a mammal are provided. This document also provides methods and materials related to stimulating immunity to folate receptors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/917,410, filed Jun. 13, 2013, which is a divisional of U.S.application Ser. No. 12/303,054 (now U.S. Pat. No. 8,486,412), filedJul. 13, 2010, which is a National Stage application under 35 U.S.C. §371 of International Application No. PCT/US2007/070237 having anInternational Filing Date of Jun. 1, 2007, which claims the benefit ofU.S. Provisional Application Ser. No. 60/810,242, filed Jun. 1, 2006.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under CA015083 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in assessingimmunity to folate receptors as well as methods and materials involvedin stimulating immunity to folate receptors.

2. Background Information

Folate receptor α (FRα) is a GPI-linked protein that is important inneurological development and is overexpressed on nearly all ovariancancers and a high proportion of breast cancers (Parker et al., Anal.Biochem., 338:284-93 (2005); Bagnoli et al., Gynecol. Oncol., 88:S140-4(2003); Holm et al., Apmis, 102:413-9 (1994); Holm et al., Biosci. Rep.,13:1-7 (1993); Holm et al., Adv. Exp. Med. Biol., 338:757-60 (1993);Weitman et al., Cancer Res., 52:3396-401 (1992); and Elnakat and Ratnam,Adv. Drug Deliv. Rev., 56:1067-84 (2004)). Overexpression of FRα □ isassociated with increased tumor aggressiveness (Toffoli et al., Int. J.Cancer, 79:121-6 (1998); Toffoli et al., Int. J. Cancer, 74:193-8(1997); Bottero et al., Cancer Res., 53:5791-6 (1993); and Campbell etal., Cancer Res., 51:5329-38 (1991)). Immunity to FRα is associated withneural tube defects in the developing embryo and cerebral folatedeficiency syndrome in children (Rothenberg et al., N. Engl. J. Med.,350:134-42 (2004); da Costa et al., Res. A. Clin. Mol. Teratol.,67:837-47 (2003); Willemsen et al., N. Engl. J. Med. 353:740 (2005);Ramaekers et al., N. Engl. J. Med., 352:1985-91 (2005); Schwartz, N.Engl. J. Med., 352:1948-50 (2005); and Ramaekers and Blau, Dev. Med.Child Neurol., 46:843-51 (2004)).

SUMMARY

This document provides methods and materials related to assessingimmunity to folate receptors. For example, this document providescompositions containing polypeptides that can be used to assess whetheror not a mammal (e.g., a mammal having cancer) has mounted an immuneresponse (e.g., T or B cell response) against a folate receptorpolypeptide (e.g., a folate receptor α). Determining whether or not acancer patient has mounted an immune response against a folate receptorpolypeptide can help clinicians assess the patient's prognosis. Forexample, a cancer patient identified as having mounted an immuneresponse against a folate receptor polypeptide can be categorized ashaving an improved prognosis as compared to a similar cancer patient whohas not mounted an immune response against a folate receptorpolypeptide. Such prognostic information can help clinicians andpatients select appropriate treatment options. Folate receptor immunityalso can be used as a marker for the early detection of cancer. Forexample, ovarian cancer usually presents as an advanced untreatabledisease. The methods and materials provided herein can be used to detectearly disease, thereby reducing mortality and morbidity.

This document also provides methods and materials related to stimulatingimmunity to folate receptors. For example, this document providescompositions containing polypeptides that can be used to stimulate animmune response against a folate receptor polypeptide. Stimulating ananti-folate receptor polypeptide response in a mammal having cancer canimprove the mammal's prognosis. For example, a cancer patient receivinga composition provided herein can mount an immune response against afolate receptor polypeptide, thereby reducing the aggressiveness of thecancer.

In general, one aspect of this document features a method for assessingFRα immunity in a mammal having cancer. The method comprises, orconsists essentially of, determining whether or not the mammal comprisesT cells reactive to an FRα polypeptide selected from the groupconsisting of FR5, FR12, FR30, FR56, FR76, FR95, FR113, FR120, FR138,FR147, FR152, FR156, FR225, and FR238. The mammal can be a human. Thecancer can be breast or ovarian cancer. The FRα polypeptide can be FR30,FR56, FR76, FR113, FR138, or FR147.

In another aspect, this document features a substantially purepolypeptide selected from the group consisting of FR5, FR12, FR30, FR56,FR76, FR95, FR113, FR120, FR138, FR147, FR152, FR156, FR225, and FR238.

In another aspect, this document features a kit comprising, orconsisting essentially of, a polypeptide selected from the groupconsisting of FR5, FR12, FR30, FR56, FR76, FR95, FR113, FR120, FR138,FR147, FR152, FR156, FR225, and FR238. The kit can comprise two or moreof the polypeptides.

In another aspect, this document features a method for increasing FRαimmunity in a mammal. The method comprises, or consists essentially of,administering a composition to the mammal under conditions effective toincrease the FRα immunity, where the composition comprises a polypeptideselected from the group consisting of FR5, FR12, FR30, FR56, FR76, FR95,FR113, FR120, FR138, FR147, FR152, FR156, FR225, and FR238. The mammalcan be a human. The composition can comprise an adjuvant.

In another aspect, this document features a method for identifying amammal as having cancer. The method comprises, or consists essentiallyof, (a) determining whether or not a mammal contains an elevated levelof anti-folate receptor polypeptide antibodies, and (b) classifying themammal as having cancer when the elevated level is present. The mammalcan be human. The cancer can be ovarian or breast cancer. The elevatedlevel of anti-folate receptor polypeptide antibodies can be a levelgreater than 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, or 50ng/mL. The anti-folate receptor polypeptide antibodies can be directedagainst at least one polypeptide selected from the group consisting ofFR5, FR12, FR30, FR56, FR76, FR95, FR113, FR120, FR138, FR147, FR152,FR156, FR225, and FR238.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of an FRα polypeptide with the distribution of MHCclass II epitopes indicated. The bottom line represents the full lengthFRα polypeptide. The numbers associated with the small lines indicatethe polypeptide epitope name. The amino acid sequence of eachpolypeptide epitope is set forth in Table 1.

FIGS. 2A-C contains three bar graphs demonstrating that a highproportion of breast and ovarian cancer patients have T cell responsesto an FRα polypeptide. Panel A is a bar graph plotting the proportionsof healthy donors and patients responding to the indicated polypeptideepitopes. Panel B is a bar graph plotting the proportions of bothovarian and breast cancer patients that responded to the indicatedpolypeptide epitopes. Panel C is a bar graph plotting the distributionof responses amongst either the amino terminus half (Amino) or thecarboxy terminus half (Carboxy) of an FRα polypeptide in both patientsand healthy donors.

FIGS. 3A-L contains graphs and images demonstrating that patients withbreast or ovarian cancer can generate immune responses to multiplepolypeptide epitopes in an FRα polypeptide. Panel A is a bar graphplotting the mean (±s.e.m.) number of spots per million peripheral bloodmononuclear cells (PBMC) for responses to phorbolmyristyl-acetate(PMA)-ionomycin, CEF (cytomegalovirus-EBV-Influenza) peptide pool, orovalbumin-derived control polypeptide (OVA) in patients and healthydonors. Panels B and C are graphs plotting the T cell frequencies (permillion PBMC) for patients and healthy donors, respectively. Each dotrepresents the mean response of three replicates for an individualspecimen. The bars for each polypeptide epitope data set represent themean T cell frequency observed for that group. Both CD4⁺ and CD8⁺ Tcells were activated in response to the polypeptide stimulation. PanelsD and E contain results of ELIspot analyses for patient 37 and healthydonor 15. Patient 37 demonstrated an FR56-specific response (183±17spots/well, mean±s.e.m., n=3) which was higher (p=0.0008) than the noantigen (26±2 spots/well) control. The responses to the CEF pool werenot significantly elevated compared to control (p>0.5). Donor 15 did notdemonstrate elevated FR56-specific T cell (3±1 spots/well, p>0.05)compared to control (5±1 spots/well), but did have an elevated CEF poolresponse (76±5 spots/well, p=0.0003). PBMC from three patients that hadresponded to FR56 were examined using IFN-γ cytokine flow cytometry.Analysis of the FR56 polypeptide using the MHCPred MHC class Ipredicting algorithm suggested this epitope contained high affinitybinding epitopes for HLA-A2 and HLA-A3. All three patients demonstrateda CD4 T cell response to the FR56 polypeptide, while two of threedemonstrated a CD8 T cell response of which a representative example isshown in FIGS. 3F-I. Panel J is a bar graph plotting the mean (±s.e.m.)number of polypeptide epitopes to which the healthy donors and ovarianor breast cancer patients responded. Panel K is a relational diagramplotting the T cell frequencies in the healthy donor and patientpopulations. Each set of data points connected by a bar represents aunique polypeptide epitope. Each data point is the mean T cell frequencycalculated within each cohort. Panel L shows that the elevated T cellfrequencies observed in the patient were mostly confined to the aminoterminus. The mean T cell frequency per patient for the amino terminuspolypeptides was 75±17 (mean±s.e.m) which, was higher than the frequencyobserved in healthy donors (24±11, p=0.007). A subsequent analysisrevealed that FR76 was a topmost predicted B cell epitope, and thispolypeptide was used to monitor for FRα-specific antibodies.

FIGS. 4A-B. Patients demonstrated increased antibody immunity to FRα.Levels of FRα-specific antibodies in patients were 68±6 ng/mL(mean±s.e.m., n=18), which was significantly higher (p<0.0001) thanlevels in the normal healthy donors (19±8 ng/mL, n=11). Antibodyresponses to TT were equivalent (p=0.3) between the two populations(patients, 30±4 μg/mL; healthy donors 27±4 μg/mL, p=0.3).

DETAILED DESCRIPTION

This document provides methods and materials related to assessingimmunity to folate receptors. For example, this document providescompositions containing polypeptides that can be used to assess whetheror not a mammal (e.g., a mammal having cancer) has mounted an immuneresponse (e.g., T or B cell response) against a folate receptorpolypeptide (e.g., a folate receptor α). A mammal having cancer that isidentified as having mounted an immune response against a folatereceptor polypeptide can be categorized as having an improved prognosisas compared to a similar mammal having cancer that has not mounted animmune response against a folate receptor polypeptide.

Immunity to a folate receptor polypeptide can be assessed in any type ofmammal. For example, humans, monkeys, cows, horses, dogs, and cats canbe assessed for the presence or absence of an immune response to afolate receptor polypeptide. Examples of folate receptor polypeptidesinclude, without limitation, folate receptor α polypeptides. An aminoacid sequence for a human folate receptor α polypeptide can be as setforth in GenBank® accession number NM_016725 (gi|9257206).

Any method can be used to assess a mammal for the presence or absence ofan immune response to a folate receptor polypeptide. For example, themethods and FRα polypeptides provided herein can be used to determinewhether or not a mammal mounted an immune response (e.g., T cellresponse) against a folate receptor α polypeptide. In some cases, amammal can be tested for the ability to respond to a panel of FRαpolypeptides. Such a panel can contain two, three, four, five, six,seven, eight, nine, ten, or more individual FRα polypeptides. Forexample, a panel can contain the FRα polypeptides set forth in Table 1.Examples of FRα polypeptides include, without limitation, FR5, FR12,FR30, FR56, FR76, FR95, FR113, FR120, FR138, FR147, FR152, FR156, FR225,and FR238. In some cases, a mammal found to have immunity to two or more(e.g., two, three, four, five, or more) FRα polypeptides within a panelcan be classified as having FRα immunity or enhanced FRα immunity, whilea mammal found to have immunity to zero or one FRα polypeptide withinthe panel can be classified as having minimal or no FRα immunity.

This document also provides substantially pure FRα polypeptides. Theterm “substantially pure” as used herein with reference to a polypeptidemeans the polypeptide is substantially free of other polypeptides,lipids, carbohydrates, and nucleic acid with which it is naturallyassociated. Thus, a substantially pure polypeptide is any polypeptidethat is removed from its natural environment and is at least 60 percentpure. A substantially pure polypeptide can be at least about 65, 70, 75,80, 85, 90, 95, or 99 percent pure. Typically, a substantially purepolypeptide will yield a single major band on a non-reducingpolyacrylamide gel.

Any method can be used to obtain a substantially pure polypeptide. Forexample, common polypeptide purification techniques such as affinitychromotography and HPLC as well as polypeptide synthesis techniques canbe used. In addition, any material can be used as a source to obtain asubstantially pure polypeptide. For example, tissue from wild-type ortransgenic animals can be used as a source material. In addition, tissueculture cells engineered to over-express a particular polypeptide ofinterest can be used to obtain substantially pure polypeptide. Further,a polypeptide can be engineered to contain an amino acid sequence thatallows the polypeptide to be captured onto an affinity matrix. Forexample, a tag such as c-myc, hemagglutinin, polyhistidine, or Flag™ tag(Kodak) can be used to aid polypeptide purification. Such tags can beinserted anywhere within the polypeptide including at either thecarboxyl or amino termini. Other fusions that could be useful includeenzymes that aid in the detection of the polypeptide, such as alkalinephosphatase.

An FRα polypeptide can be obtained recombinantly, synthetically, orcommercially. An FRα polypeptide can have a non-naturally occurringsequence or can have a sequence present in any species (e.g., human,rat, or mouse). In some cases, an FRα polypeptide can contain one ormore amino acid analogs or other peptidomimetics. The subunits of an FRαpolypeptide may be linked by peptide bonds or other bonds such as, forexample, ester or ether bonds. An FRα polypeptide can be a full-lengthFRα polypeptide, a precursor FRα polypeptide, or a fragment of afull-length FRα polypeptide.

In some cases, an FRα polypeptide can contain one or more modifications.For example, an FRα polypeptide can be modified to be pegylated or tocontain additional amino acid sequences such as an albumin sequence(e.g., a human albumin sequence). In some cases, an FRα polypeptide canbe a fusion polypeptide, such as a fusion polypeptide that contains afragment of an albumin sequence. In some cases, an FRα polypeptide canbe covalently attached to oligomers, such as short, amphiphilicoligomers that enable oral administration or improve the pharmacokineticor pharmacodynamic profile of a conjugated FRα polypeptide. Theoligomers can comprise water soluble polyethylene glycol (PEG) and lipidsoluble alkyls (short chain fatty acid polymers). See, for example,International Patent Application Publication No. WO 2004/047871. In somecases, an FRα polypeptide can be fused to the Fc domain of animmunoglobulin molecule (e.g., an IgG1 molecule) such that activetransport of the fusion polypeptide occurs across epithelial cellbarriers via the Fc receptor. In some cases, an FRα polypeptide can be afusion polypeptide, such as a fusion polypeptide that contains an FRαpolypeptide fused to an immunogenic polypeptide. In some cases, an FRαpolypeptide can be designed to contain foreign T-cell epitopes so thatadministration of the polypeptide to a mammal produces or increasesimmunity to a folate receptor polypeptide in the mammal.

Any method can be used to obtain a folate receptor polypeptide (e.g., anFRα polypeptide). For example, molecular cloning techniques can be usedto prepare a nucleic acid construct encoding a folate receptorpolypeptide. Such a construct can be expressed in an organism such as E.coli or S. cerevisiae, or in a cell line, for example, and the expressedpolypeptide can be purified from cellular extracts or from culturesupernatants. A folate receptor polypeptide also can be chemicallysynthesized.

This document also provides methods and materials for increasing amammal's immunity to a folate receptor polypeptide. For example, one ormore of the FRα polypeptides provided herein can be formulated into acomposition that can be administered to a mammal (e.g., a mouse, a rat,a cat, a dog, a horse, a cow, a non-human primate such as a cynomolgusmonkey, or a human) under conditions that lead to increased immunityagainst an FRα polypeptide. Such composition can include ingredientsfound in vaccines such as adjuvants. For example, a composition providedherein can contain one or more FRα polypeptides in combination with anadjuvant (e.g., aluminum hydroxide, aluminum phosphate, calciumphosphate, monophosphoryl lipid A, an ISCOM with Quil-A, or a Syntexadjuvant formulations (SAF)® containing a threonyl derivative or muramyldipeptide). In some cases, an FRα polypeptide can contain a sequencecapable of generating a CD4⁺ T cell response, a CD8⁺ T cell response, orboth.

Alum as well as other aluminum-based compounds (e.g., Al₂O₃) can becombined with a folate receptor polypeptide (e.g., an FRα polypeptide)to form a composition that elicits an immune response against a folatereceptor polypeptide (e.g., an FRα polypeptide) when administered to amammal. Aluminum-based compounds can be obtained from various commercialsuppliers. For example, REHYDRAGEL® adjuvants can be obtained fromReheis Inc. (Berkeley Heights, N.J.). REHYDRAGEL® adjuvants are based oncrystalline aluminum oxyhydroxide, and are hydrated gels containingcrystalline particles with a large surface area (about 525 m²/g). TheirAl₂O₃ content typically ranges from about 2 percent to about 10 percent.Rehydragel LG, for example, has an Al₂O₃ content of about 6 percent, andflows readily upon slight agitation. Rehydragel LG also has a proteinbinding capacity of 1.58 (e.g., 1.58 mg of bovine serum albumin boundper 1 mg of Al₂O₃), a sodium content of 0.02 percent, a chloride contentof 0.28 percent, undetectable sulphate, an arsenic level less than 3ppm, a heavy metal content less than 15 ppm, a pH of 6.5, and aviscosity of 1090 cp. Rehydragel LG can be combined with a polypeptidesolution (e.g., a polypeptide in PBS) to yield Al(OH)₃. In addition,ALHYDROGEL™, an aluminum hydroxy gel adjuvant, (Alhydrogel 1.3%,Alhydrogel 2.0%, or Alhydrogel “85”) obtained from Brenntag StinnesLogistics can be used.

MN51 also can be combined with a folate receptor polypeptide (e.g., anFRα polypeptide) to form a composition that elicits an immune responseagainst a folate receptor polypeptide (e.g., an FRα polypeptide) whenadministered to a mammal. MN51 (MONTANIDE® Incomplete SEPPIC Adjuvant(ISA) 51) can be obtained from Seppic (Paris, France). Other adjuvantsinclude immuno-stimulating complexes (ISCOMs) that can contain suchcomponents as cholesterol and saponins. Adjuvants such as FCA, FIA,MN51, MN720, and Al(OH)₃ are commercially available from companies suchas Seppic, Difco Laboratories (Detroit, Mich.), and Superfos BiosectorA/S (Vedbeak, Demark).

In some cases, a composition provided herein can contain one or moreadditional immunostimulatory components. These include, withoutlimitation, muramyldipeptide (e.g.,N-acetylmuramyl-L-alanyl-D-isoglutamine; MDP), monophosphoryl-lipid A(MPL), and formyl-methionine containing tripeptides such asN-formyl-Met-Leu-Phe. Such compounds are commercially available fromSigma Chemical Co. (St. Louis, Mo.) and RIBI ImmunoChem Research, Inc.(Hamilton, Mont.), for example.

A “unit dose” of a composition refers to the amount of a compositionadministered to a mammal at one time. A unit dose of the compositionsprovided herein can contain any amount of polypeptide. For example, aunit dose of a composition can contain between about 10 μg and about 1 g(e.g., 10 μg, 15 μg, 25 μg, 30 μg, 50 μg, 100 μg, 250 μg, 280 μg, 300μg, 500 μg, 750 μg, 1 mg, 10 mg, 15 mg, 25 mg, 30 mg, 50 mg, 100 mg, 250mg, 280 mg, 300 mg, 500 mg, 750 mg, or more) of a polypeptide. In someembodiments, the polypeptide can be dissolved or suspended in aphysiological buffer such as, for example, water or phosphate bufferedsaline (PBS), pH 7.0. The solution of polypeptide then can be combinedwith the adjuvant and any other components of the composition.

Similarly, a unit dose of a composition can contain any amount of anadjuvant. For example, a unit dose can contain between about 10 μL andabout 1 mL (e.g., 10 μL, 25 μL, 50 μL, 100 μL, 250 μL, 500 μL, 750 μL,800 μL, 900 μL, or 1 mL) of one or more adjuvants. In addition, a unitdose of a composition can contain any amount of anotherimmunostimulatory component. For example, a composition provided hereincan contain between about 10 μg and about 1 g (e.g., 10 μg, 15 μg, 25μg, 30 μg, 50 μg, 100 μg, 250 μg, 280 μg, 300 μg, 500 μg, 750 μg, 1 mg,10 mg, 15 mg, 25 mg, 30 mg, 50 mg, 100 mg, 250 mg, 280 mg, 300 mg, 500mg, 750 mg, or more) of an immunostimulatory component.

The compositions provided herein can contain any ratio of adjuvant topolypeptide. The adjuvant:antigen ratio can be 50:50 (vol:vol), forexample. Alternatively, the adjuvant:antigen ratio can be, withoutlimitation, 90:10, 80:20, 70:30, 64:36, 60:40, 55:45, 40:60, 30:70,20:80, or 90:10.

This document also provides methods for preparing the compositionsprovided herein. Such methods can involve suspending an amount of apolypeptide (e.g., 100 μg of an FRα polypeptide) in a suitable amount ofa physiological buffer (e.g., 50 μL of PBS pH 7.0), and then combiningthe suspended or dissolved polypeptide with a suitable amount of anadjuvant (e.g., 50 μL of MN51 or 100 μL of REHYDRAGEL®). The combiningstep can be achieved by any method, including stirring, shaking,vortexing, or passing back and forth through a needle attached to asyringe, for example. It is noted that the composition can be preparedin batch such that enough unit doses are obtained for multipleinjections (e.g., injections into multiple mammals or multipleinjections into the same mammal).

In general, compositions containing an FRα polypeptide can be used as avaccine to induce or increase immunity against a folate receptorpolypeptide (e.g., an endogenous FRα polypeptide) in a mammal (e.g., amammal having cancer). As described herein, administering a compositioncomprising an FRα polypeptide to a mammal having cancer can induce orincrease an immune response (e.g., a T cell response) against a folatereceptor polypeptide in the mammal, which, in turn, can reduce theaggressiveness of a cancer in the mammal and improve the mammal'sprognosis. In some cases, administering a composition comprising an FRαpolypeptide to a mammal that is susceptible to developing cancer (e.g.,a mammal that has a family history of cancer) can induce or increase animmune response against a folate receptor polypeptide in the mammalwhich, in turn, can delay or prevent the onset of cancer or reduce theaggressiveness of a cancer that develops in the mammal.

The compositions provided herein can be administered by a number ofmethods. Administration can be, for example, topical (e.g., transdermal,ophthalmic, or intranasal); pulmonary (e.g., by inhalation orinsufflation of powders or aerosols); oral; or parenteral (e.g., bysubcutaneous, intrathecal, intraventricular, intramuscular, orintraperitoneal injection, or by intravenous drip). Administration canbe rapid (e.g., by injection) or can occur over a period of time (e.g.,by slow infusion or administration of slow release formulations).

Any dose can be administered to a mammal. Dosages can vary depending onthe relative potency of individual compositions, and can generally beestimated based on data obtained from in vitro and in vivo animalmodels. Typically, a dosage is from about 0.01 μg to about 100 g per kgof body weight, and may be given once or more daily, weekly, monthly,yearly, or less often. Following successful administration, it may bedesirable to have the subject undergo additional booster administrationsto maintain a suitable level of the immune response.

The immune response to a folate receptor polypeptide produced in amammal by administration of a composition provided herein can beassessed using any appropriate method. For example, the titer ofanti-folate receptor antibodies can be measured. In addition, a “titerdilutions₅₀ value” can be determined by using an ELISA (e.g., with oneor more immobilized FRα polypeptides) and measuring the optical density(OD) of dilutions (e.g., serial dilutions) of a serum sample from amammal. The dilution factor that results in a 50 percent reduction fromthe maximal OD is considered to be the titer dilutions₅₀ value. Thisvalue can be calculated by curve fitting using, for example, theSOFTmax® Pro 4.0 software program that is available from MolecularDevices, Inc. (Sunnyvale, Calif.). Using a four parameter non-linearregression for curve fitting, this program can be used to fit datapoints to a curve and determine the titer dilutions₅₀ value. In somecases, PBMCs from a mammal can be analyzed using an ELIspot to detect Tcells reactive to FRα polypeptides as described herein. In some cases,an antibody ELISA assay or cytokine flow cytometry can be performed asdescribed herein to assess immunity against a folate receptorpolypeptide in a mammal following administration of a compositioncomprising an FRα polypeptide.

This document also provides methods and materials for identifying amammal (e.g., a human) as having cancer (e.g., breast or ovariancancer). For example, a mammal can be identified as having cancer if thelevel of anti-folate receptor polypeptide antibodies in the mammal(e.g., in a serum sample from the mammal) is an elevated level. If thelevel of anti-folate receptor polypeptide antibodies in a mammal (e.g.,in a serum sample from the mammal) is not an elevated level, then themammal can be classified as not having cancer.

An anti-folate receptor polypeptide antibody can be any antibody thatbinds to a folate receptor polypeptide. For example, an anti-folatereceptor polypeptide antibody can be an antibody that binds to an FRαpolypeptide. In some cases, an anti-folate receptor polypeptide antibodycan be an antibody directed against a polypeptide selected from thegroup consisting of FR5, FR12, FR30, FR56, FR76, FR95, FR113, FR120,FR138, FR147, FR152, FR156, FR225, and FR238. An anti-folate receptorpolypeptide antibody can bind to a folate receptor polypeptide at anaffinity of at least 10⁴ mol⁻¹ (e.g., at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰, 10¹¹, or 10¹² mol⁻¹). In addition, an anti-folate receptorpolypeptide antibody can be of any type, (e.g., IgG, IgM, IgD, IgA orIgY), class (e.g., IgG1, IgG4, or IgA2), or subclass.

The term “elevated level” as used herein with respect to the level ofanti-folate receptor polypeptide antibodies is any level that is greaterthan a reference level for anti-folate receptor polypeptide antibodies.The term “reference level” as used herein with respect to anti-folatereceptor polypeptide antibodies is the level of anti-folate receptorpolypeptide antibodies found in mammals free of cancer. For example, areference level of anti-folate receptor polypeptide antibodies can bethe average level of anti-folate receptor polypeptide antibodies that ispresent in samples obtained from a random sampling of 20 or more (e.g.,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,100, or more) healthy mammals. It will be appreciated that levels fromcomparable samples are used when determining whether or not a particularlevel is an elevated level.

An elevated level of anti-folate receptor polypeptide antibodies can beany level provided that the level is greater than a correspondingreference level for anti-folate receptor polypeptide antibodies. Forexample, an elevated level of anti-folate receptor polypeptideantibodies can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or moretimes greater than the reference level for anti-folate receptorpolypeptide antibodies. In addition, a reference level can be anyamount. For example, a reference level for anti-folate receptorpolypeptide antibodies can be zero. In this case, any level ofanti-folate receptor polypeptide antibodies greater than zero would bean elevated level. In some cases, an elevated level of anti-folatereceptor polypeptide antibodies can be a level greater than about 25ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 55 ng/mL, or 60ng/mL.

Any method can be used to determine the level of anti-folate receptorpolypeptide antibodies present within a mammal. For example, the levelof anti-folate receptor polypeptide antibodies present within a samplefrom the mammal can be determined using FRα polypeptides providedherein. One or more than one FRα polypeptide can be immobilized on asurface (e.g., the surface of an ELISA plate), non-specific binding canbe blocked, and the immobilized polypeptides can be incubated with asample (e.g., a sample of serum) from a mammal. A labeled antibody thatcan bind to an anti-folate receptor polypeptide antibody can be used todetect a level of anti-folate receptor polypeptide antibodies bound tothe one or more than one FRα polypeptide. An antibody can be labeleddirectly or indirectly. Suitable labels include, without limitation,radioisotopese (e.g., ¹²⁵I, ¹³¹I, ³⁵S, ³H, ³²P, ³³P, or ¹⁴C),fluorophores (e.g., fluorescein, fluorescein-5-isothiocyanate (FITC),PerCP, rhodamine, or phycoerythrin), luminescent moieties (e.g., Qdot™nanoparticles supplied by the Quantum Dot Corporation, Palo Alto,Calif.), compounds that absorb light of a defined wavelength, or enzymes(e.g., alkaline phosphatase or horseradish peroxidase). Antibodies canbe indirectly labeled by conjugation with biotin and then detected withavidin or streptavidin labeled with a molecule described above. Methodsof detecting or quantifying a label depend on the nature of the labeland are known in the art. Examples of detectors include, withoutlimitation, x-ray film, radioactivity counters, scintillation counters,spectrophotometers, colorimeters, fluorometers, luminometers, anddensitometers. Combinations of these approaches (including “multi-layer”assays) familiar to those in the art can be used to enhance thesensitivity of assays.

In some cases, a level of anti-folate receptor polypeptide antibodiescan be determined in a sample (e.g., a serum sample) from a mammal byexploiting the phenomenon of surface plasmon resonance, which results ina change in the intensity of surface plasmon resonance upon binding thatcan be detected qualitatively or quantitatively by an appropriateinstrument, e.g., a Biacore apparatus (GE Healthcare, United Kingdom).One or more than one FRα polypeptide can be immobilized on the sensorsurface of a Biacore apparatus and the immobilized polypeptide can beincubated with a sample (e.g., a diluted serum sample) from a mammal todetermine a level of anti-folate receptor polypeptide antibodies. Astandard curve using known quantities of anti-folate receptorpolypeptide antibodies can be generated to aid in the quantitation ofanti-folate receptor polypeptide antibody levels.

Any type of sample can be used to evaluate a level of anti-folatereceptor polypeptide antibodies including, without limitation, serum,blood, and plasma. In addition, any method can be used to obtain asample. For example, a blood sample can be obtained by peripheralvenipuncture. Once obtained, a sample can be manipulated prior tomeasuring the level of anti-folate receptor polypeptide antibodies. Forexample, a blood sample can be heparanized, centrifuged, or frozen priorto analysis. In addition, replicates and multiple dilutions of a samplecan be analyzed.

This document also provides kits that can be used to perform a methodprovided herein (e.g., to assess immunity against a folate receptorpolypeptide in a mammal having cancer). Such kits can include FRαpolypeptides, labeled secondary antibodies, control serums (e.g., serumsthat do or do not contain antibodies directed against, or T cellsreactive to, folate receptor polypeptides), ELISA plates, or dataanalysis software, optionally together with any other appropriatereagent, tool, or instruction for performing a method described herein.Appropriate informational material can be descriptive, instructional,marketing, or other material that relates to the methods describedherein and/or the use of the reagents for the methods described herein.For example, the informational material can relate to assessing orincreasing immunity against a folate receptor polypeptide in a mammal.In addition, the informational material of a kit can be contactinformation, e.g., a physical address, e-mail address, website, ortelephone number, where a user of the kit can obtain substantiveinformation about analyzing immunity against a folate receptorpolypeptide and interpreting the results, particularly as they apply toprognosis of a human having cancer.

The informational material of the kits can be in any form. In manycases, the informational material, e.g., instructions, can be providedin printed matter, e.g., a printed text, drawing, and/or photograph,e.g., a label or printed sheet. Informational material can be providedin other formats, such as Braille, computer readable material, videorecording, or audio recording. Informational material can also beprovided in any combination of formats.

The kit can include one or more containers for the reagents forassessing or increasing immunity, such as reagents for performing flowcytometry, an ELISA, or any other method described herein. The kit cancontain separate containers, dividers, or compartments for the reagentsand informational material. A container can be labeled for use for thediagnosis and/or prognosis of a human relating to the development andtreatment of cancer.

This document also provides methods and materials to assist medical orresearch professionals in determining whether or not a mammal hascancer, or in determining whether or not a mammal having cancer has apoor prognosis. Medical professionals can be, for example, doctors,nurses, medical laboratory technologists, and pharmacists. Researchprofessionals can be, for example, principle investigators, researchtechnicians, postdoctoral trainees, and graduate students. Aprofessional can be assisted by (1) determining the presence, absence,or level of immunity against a folate receptor polypeptide, and (2)communicating information about the presence, absence, or level to thatprofessional.

Any appropriate method can be used to communicate information to anotherperson (e.g., a professional). For example, information can be givendirectly or indirectly to a professional. In addition, any type ofcommunication can be used to communicate the information. For example,mail, e-mail, telephone, and face-to-face interactions can be used. Theinformation also can be communicated to a professional by making thatinformation electronically available to the professional. For example,the information can be communicated to a professional by placing theinformation on a computer database such that the professional can accessthe information. In addition, the information can be communicated to ahospital, clinic, or research facility serving as an agent for theprofessional.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Assessing Folate Receptor Immunity

Materials: Phorbol myristate acetate (PMA), human serum albumin (HSA),polyclonal human IgG, tetanus toxin (TT), and ionomycin were from Sigma(St. Louis, Mo., USA). Goat anti-human horseradish peroxidase(HRP)-conjugated antibody was obtained from Santa Cruz Biotechnology(Santa Cruz, Calif.). FITC-conjugated anti-CD4, PE-conjugatedanti-IFN-γ, APC-conjugated anti-CD8, and all cytokine flow cytometryreagents were obtained from BD Biosciences (San Jose, Calif.). Hank'sbalanced salts solution (HBSS), RPMI-1640 and phosphate-buffered salinewere from Cellgro (Hernden, Va., USA). Ficoll-Paque was from AmershamBiosciences (Uppsala, Sweden). The CEF viral polypeptide pool was fromthe NIH AIDS Research and Reference Reagent Program.

Epitope Prediction and Synthesis: FRα epitopes were predicted asdescribed elsewhere (Knutson and Disis, Cancer Immunol. Immunother.,54:721-8 (2005)) using RANKPEP (World Wide Web at mifoundation.org)(Table 1, FIG. 1). The sequence for FRα was from the Entrez Database(accession #P15328). Polypeptides were predicted that potentially boundto HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR7, and HLA-DR11(Table 1). The algorithm predicted polypeptides of fifteen residues. Ifa polypeptide was predicted to bind to at least three different HLA-DRalleles, it was selected. In some cases, a polypeptide was extendedbeyond fifteen residues so that a predicted binding core was flanked byat least three amino acids. The polypeptides, 15-19 residues, wereproduced to >95% purity by HPLC and mass-spectrometry at the MayoClinic. Routine testing of polypeptides demonstrated that they arenegative for contamination by splenocyte blastogenesis assays. In selectcases, the predicted epitopes were further analyzed using MHCPred (WorldWide Web at jenner.ac.uk) for encompassed HLA-A1 (0101), -A2 (0102), and-A3 (0301). These three HLA-A alleles were selected because they arethose which are the highest amongst Caucasians, African Americans, andHispanics. Polypeptides were considered to potentially contain embeddedHLA-class I epitopes if the algorithm predicted regions with IC₅₀ valuesof less than or equal to 500 nM. Additionally, FRα-derived T cellepitopes were also analyzed using B cell epitope prediction algorithmsABCpred (www.imtech.res.in) and Antigenic (bioinfo.bgu.ac.il), todetermine if they would be useful for detecting antibodies.

Patients and Donors: Ten healthy donor samples and twenty patientsamples were obtained at the Mayo Clinic. Ten patient samples wereobtained from the University of Washington (shipped on dry ice) and wereprocessed and stored using the same procedures as the Mayo Clinicsamples. Patients were free from active treatment for at least 30 dayswhen blood was drawn. Blood (200 mL) was collected over about a sevenmonth period. The mean (±s.e.m) ages of the healthy donors and patientswere 42±11 and 55±2, respectively (p<0.0001). Healthy donors wererecruited by means of local advertisement which gave the details of theblood draw. For the antibody ELISA studies, sera was available fromeleven healthy controls and nineteen patients.

Peripheral Blood Mononuclear Cell Preparation (PBMC): An equal volume ofHank's balanced salt solution (HBSS) was added to the blood samples.Twenty-five mL of diluted blood was overlaid onto 15 mL of Ficoll-Paquein a 50 mL conical test tube centrifuged at 374×g for 35 minutes at roomtemperature. Following centrifugation, the buffy coats were centrifuged(12 minutes, 216×g). The cell pellets were washed and resuspended infreezing media (RPMI supplemented with 12.5 HSA, penicillin,streptomycin, and 2 mM glutamine). Cells were adjusted to 20×10⁶/mLcells, and an equal volume of cold 12.5% HSA RPMI containing 25% DMSO(Sigma D2650) was added. Cells were aliquoted and transferred to a −80°C. freezer. After overnight incubation, the vials were transferred toliquid nitrogen.

For in vitro assay, each vial of cells was thawed, and the contents weretransferred to a test tube containing 12 mL of cell culture media.Following centrifugation, the cells were resuspended in 2-3 mL ofmedium, and viability was determined by dye exclusion. The mean numberof PBMC purified from the blood of donors was 1.6±0.07×10⁶ cells/mL ofwhole blood for healthy donors and 1.0±0.07×10⁶/mL for cancer patients(p<0.0001). This cryopreservation and thawing procedure was optimizedfor recovery of antigen-specific T cell function (Disis et al., J.Immunol. Methods, 308(1-2):13-8 (2006)).

IFN-γ ELISpot Analysis: A 10-day ELIspot for detecting low-frequency Tcells was used to determine reactivity to the FRα-derived polypeptides(Table 1). ELIspots were carried out in groups of two donors (twohealthy donors, one healthy donor/one cancer patient, or two cancerpatients). The assay was carried out as described elsewhere (Knutson etal., J. Clin. Invest., 107:477-84 (2001)). On day 1, 2.5×10⁵ PBMCs/wellwere plated into 96-well plates in 3-well replicates in 200 μL ofRPMI-1640 containing L-glutamine, penicillin, streptomycin, and 10%fetal calf serum (T-cell medium) in the presence or absence of 10 μg/mLpolypeptide antigen. The cells were incubated at 37° C. and IL-2(Zeptometric, Inc., Buffalo, N.Y.) was added to 10 U/mL on day 5. On day8, 2.5×10⁵/well irradiated autologous PBMCs and 10 μg/mL antigens wereadded. On day 9, the cells were transferred to an anti-IFN-γ-coatednitrocellulose (NC)-plate (Millipore Corporation, Bedford, Mass.). TheNC-plate was incubated (37° C.) for a further 20-24 hours followed bywashing three times using PBS containing 0.05% Tween-20. The plate wasthen incubated for 2.5 hours at RT in PBS with 5 μg/mL biotinylatedanti-IFN-γ Ab, washed in PBS, and further incubated with 100 μL/wellavidin-horseradish peroxidase (HRP, Vector Laboratories, Burlingame,Calif.)) for 2 hours at room temperature. After three washes in PBS, theplate was incubated with 100 μL/well HRP-colorimetric substrate (VectorLaboratories) for 20-30 minutes, rinsed with cool tap water, and allowedto dry completely. The nitrocellulose plates were read on an AID ELIspotreader (Cell Technology, Inc., Columbia, Md.; reader software v.3.1.1.).A positive response was defined as a frequency that was significantly(p<0.05, two-tailed t test) greater than the mean of control no-antigenwells and detectable (i.e., >1:100,000). A 17-amino acid polypeptide(KISQAVHAAHAEINEAG; SEQ ID NO:1) derived from chicken egg ovalbumin,produced in the same fashion as the FRα polypeptides, was used as acontrol polypeptide. This polypeptide was predicted to bind to HLA-DR2and DR5 but not DR1, 3, 4, 7, or 11. The anti-IFN-γ and biotinylatedanti-IFN-γ antibody pair were obtained from Mabtech (Sweden).

TABLE 1 FRα polypeptides SEQ ID LENGTH # OF SEQUENCE NO: POSITIONSDESIGNATION (AA) DR1 DR2 DR3 DR4 DR5 DR7 DR11 HLA MTTQLLLLLVWVAVVGEAQ 2 5-23 FR5  19 X X X X X 5 LLVWVAVVGEAQTRI 3 12-26 FR12  15 X X X 3RTELLNVCMNAKHHKEK 4 30-46 FR30  17 X X X 3 QCRPWRKNACCSTNT 5 56-70 FR56 15 X X X 3 KDVSYLYRFNWNHCGEMA 6 76-93 FR76  18 X X X X X X 6ACKRHFIQDTCLYECS 7  95-110 FR95  16 X X X 3 LGPWIQQVDQSWRKERV 8 113-129FR113 17 X X X X X 5 VDQSWRKERVLNVPL 9 120-134 FR120 15 X X X X X 5DCEQWWEDCRTSYTCK 10 138-153 FR138 16 X X X 3 RTSYTCKSNWHKGWNWT 11147-163 FR147 17 X X X 3 CKSNWHKGWNWTSGFN 12 152-167 FR152 16 X X X X X5 WHKGWNWTSGFNKCAVGA 13 156-174 FR156 18 X X X 3 VARFYAAAMSGAGPWA 14225-240 FR225 16 X X X X 4 PWAAWPFLLSLALMLLWL 15 238-255 FR238 18 X X XX X 5

Antibody Enzyme-Linked Immunosorbent Assay (ELISA): Antigen (10 μg/well)was prepared in 0.06 M carbonate buffer and added to ELISA microtiterplates for 24 hours. Plates were washed with PBS and blocked with 3%BSA-PBS. One hundred microliters of diluted serums (1:125 forpolypeptide and 1:40 for tetanus toxoid in 1% BSA-PBS) were added, andthe plates were further incubated for 2 hours at room temperaturefollowed by washing with PBS/0.1% Tween-20. A 1:2000 dilution ofanti-IgG-HRP was added to wells for 1 hour followed by washing and colordevelopment with TMB (3,3′,5,5′ tetramethylbenzidine substrate was added(100 μL) to the wells. Color development was stopped with 50 μL of a 0.1N HCl solution. For the standard curve, serial dilutions of human IgGwere added to separate wells. As a control, a polypeptide derived fromhuman collagen II, HII.71 (PPGLTGPAGEPGRQGSPGAD; SEQ ID NO:16), wasused.

Cytokine Flow Cytometry: PBMC were cultured with peptide (10 μg/mL) forseven days. IL-2 (20 U/mL) were added on Day 3. After seven days, thecells were distributed into a 96-well plate with fresh irradiatedautologous PBMC. Either medium alone or medium supplemented with PHA-L(20 μg/well) or recall polypeptide (10 μg/well) was added to theappropriate wells for 29 hours. Golgi-Stop was added for the last fivehours followed by washing in PBS-0.5% BSA. The cells were resuspended inthe same buffer containing anti-CD4-FITC and anti-CD8-APC for 30minutes, followed by washes and fixing. The cells were permeabilizedalone or with unconjugated anti-human IFN-γ (adsorption control)followed by washing and incubation with anti-human anti-IFN-γ-APC for 30minutes. The cells were washed, fixed, and analyzed using a BDBiosciences FACscan flow cytometer and CellQuest Pro Software (version4.0.2., BD Biosciences). A response to antigen was considered aspositive if there was at least a 50% increase in IFN-γ□ cells, and itwas blocked by the adsorption control.

Statistics: The T cell magnitude for each donor was summed across all 14polypeptides which, along with the response multiplicity, was comparedwith age using regression. Student's t test was used for means unlessthe data were not normally distributed, in which case Mann-Whitney testwas used. Fisher's Exact Test was used for comparing proportions. Aproportion was considered elevated, relative to other polypeptides, ifthat proportion was statistically elevated relative to the meanproportion, 8.7%, which is the ratio of the total number of significant(p<0.05, two-tailed t test) polypeptide-specific responses over thetotal number of donors. Tests were performed using InStat (v.3.00),GraphPad Software (San Diego, Calif.). Changes were consideredsignificant if p<0.05. Unless specified, one-tailed tests were used. Themean proportion method (Table 2) is used because the ELIspot does notprovide a continuous read out due to (1) the limits of detection, and(2) zero value assignment if not significantly different than control.The use of a mean proportion is a rigorous modification of a techniquethat is used in prior immunologic studies.

TABLE 2 Fisher's Exact Test for Proportion Responding Healthy DonorPatients Proportion Peptide Proportion (%) (%) p Value* p Value** FR5 617 0.2 0.2 FR12 6 7 0.6 0.7 FR30 6 30 0.02 0.04 FR56 6 33 0.007 0.03FR76 11 13 0.4 0.6 FR95 6 23 0.07 0.1 FR113 0 27 0.03 0.02 FR120 11 130.4 0.6 FR138 22 30 0.02 0.4 FR147 22 40 0.001 0.2 FR152 6 23 0.07 0.1FR156 17 23 0.07 0.4 FR225 22 7 0.6 0.1 FR238 0 23 0.07 0.03 *Fisher'sExact Test (one-tailed) comparing if the proportion of patientsresponding to polypeptide is higher than the mean proportion of 8.7%.**Fisher's Exact Test comparing if the proportion of patients is higherthan the proportion of healthy individuals responding to polypeptide.Those comparisons deemed statistically significant are bolded.

Patients with breast and ovarian cancer generate immunity to multiplepolypeptide epitopes in FRα. The FRα polypeptides predicted to beimmunogenic were distributed throughout the receptor (FIG. 1). Responsesto PMA/ionomycin, the CEF polypeptide pool, and an ovalbumin-derivedcontrol polypeptide were not different between the two populations (FIG.3A). T cell frequencies were determined for each of the FRα polypeptidesfor the patients (FIG. 3B) and the healthy donors (FIG. 3C). The meanfrequencies for each of the polypeptides ranged from 0-124 Tcells/million PBMC for the normals and 1-162/million PBMC for thepatients. The overall mean FRα-specific T cell frequency, consideringall polypeptides, for patients was 74±11 (mean±s.e.m., n=448) and forhealthy donors was 46±10 (n=226) (p=0.05). An ELIspot for patient 37 andhealthy donor 15 was performed (FIGS. 3D-E). Patient 37 demonstrated anFR56-specific response (183±17 spots/well, mean±s.e.m., n=3) which washigher (p=0.0008) than no antigen (26±2 spots/well) control. Theresponses to the CEF pool were not significantly elevated compared tocontrol (p>0.5). Donor 15 did not demonstrate elevated FR56-specific Tcell (3±1 spots/well, p>0.05) compared to control (5±1 spots/well), butdid have an elevated CEF pool response (76±5 spots/well, p=0.0003).

Both CD4⁺ and CD8⁺ T cells were activated in response to the polypeptidestimulation. PBMC from three patients that had responded to FR56 wereexamined using IFN-γ cytokine flow cytometry. Analysis of the FR56polypeptide using the MHCPred MEW class I predicting algorithm suggestedthis epitope contained high affinity binding epitopes for HLA-A2 andHLA-A3. All three patients demonstrated a CD4 T cell response to theFR56 polypeptide while two of three demonstrated a CD8 T cell responseof which a representative example is shown in FIGS. 3F-I.

It was observed that ovarian and breast cancer patients demonstratedimmunity to 3±0.6 (mean±s.e.m.) and 3±0.7 FRα-derived polypeptides,respectively (FIG. 3J). These levels of reactivity were higher thanhealthy donors who responded to 1±0.5 (n=18) polypeptides. As shown inthe relational diagram (FIG. 3K), the calculated frequency of eleven ofthe polypeptides was increased in patients while three were similar ordecreased. The elevated T cell frequencies observed in the patient weremostly confined to the amino terminus as shown in FIG. 3L. The mean Tcell frequency per patient for the amino terminus polypeptides was 75±17(mean±s.e.m) which was higher than the frequency observed in healthydonors (24±11, p=0.007). There was no difference in the mean T cellprecursor frequencies to the carboxy polypeptides (p=0.1). Further,there were no associations between the T cell frequencies and the numberof HLA-DR alleles to which each polypeptide was predicted to bind. Therewere no effects of age (range 25-73) on either the numbers ofpolypeptides that elicited an immunogenic response (r²=0.006, p=0.6,2-tailed) or the magnitude of the T cell responses (r²=0.004, p=0.7).

A high proportion of breast and ovarian cancer patients have t cellresponses to FRα. The percentage of patients who responded to eachpolypeptide was determined and found to range from 7-40% (FIG. 2A).Patients responded in higher proportions (p<0.05) to FR30 (30%), FR56(33%), FR113 (27%), FR138 (30%), and FR147 (40%) (Table 2). Responseswere more frequently observed in cancer patients than in healthy donors.Of the 14 polypeptides, four (FR30, FR56, FR113, FR238) generatedresponses in more patients than healthy donor counterparts (Table 2).Reactivity to three of these, FR30, FR56, and FR113, was observed ingreater than 25% of patients. Of the fourteen FRα polypeptides, FR56 wasrecognized more often (p=0.05) by ovarian cancer patients than by breastcancer patients (FIG. 2B). Overall, 69% and 76% of ovarian and breastcancer patients, respectively, demonstrate immunity to at least oneeptiope of FRα.

The responses in the patients were equally distributed amongst the aminoterminus polypeptides (FR5-FR113) and the carboxy terminus polypeptides(FR120-FR238). 47% and 53% of the polypeptide responses were directedtoward the amino and carboxy terminus polypeptides, respectively (p=0.5;FIG. 2C). The responses observed in normal control individuals were morefrequently observed in the carboxy terminus (72%) compared to the aminoterminus (28%).

Breast and ovarian cancer patients demonstrate FRα-specific antibodyimmunity. Patients demonstrated increased antibody immunity to FRα (FIG.4A). Levels of FRα-specific antibodies in patients were 68±6 ng/mL(mean±s.e.m., n=18), which was significantly higher (p<0.0001) thanlevels in the normal healthy donors (19±8 ng/ml, n=11). Antibodyresponses to TT were equivalent (p=0.3) between the 2 populations(patients, 30±4 μg/mL; healthy donors 27±4 μg/mL, p=0.3; FIG. 4B).

The detection of pre-existent immunity to cancer antigens is usefulbecause it identifies antigens to which tolerance induction by the hostis nonexistent, incomplete or reversible. Furthermore, this immunity canindicate that a patient's immune system may have responded to the tumorand is potentially involved in tumor rejection. These antigens could betargeted with immune-based cancer treatment and prevention strategiessuch as cancer vaccines as it may be easier to expand a memory pool of Tcells as compared to generating new immunity. As described herein, anMHC class II algorithm was used to define immunogenic regions of theFRα, and FRα-specific immunity was found to be prevalent in patientswith breast or ovarian cancer. Of the 14 putative MHC class IIpolypeptides identified, five polypeptides were recognized by greaterthan 25% of patients. The response proportion to three of thesepolypeptides was higher than that observed in a healthy volunteer donorpopulation. Overall, patients responded to an average of threeFRα-derived polypeptides suggesting a multi-epitope response whereas thehealthy donors responded to one polypeptide and at a lower T cellfrequency. Lastly, immunity to the FRα in cancer patients targeted boththe amino and carboxy-terminal halves of the molecule whereas immunityobserved in healthy donors largely targeted the carboxy terminal half.Collectively, these results demonstrate that tolerance to the FRα isminimal and the majority of patients have immunity to multiple epitopes.

The results provided herein demonstrate that immunity to FRα isprevalent in breast and ovarian cancer patients. Immunity to tumorantigens is typically thought to be very low or undetectable for breastand ovarian cancer. However, this understanding is limited by thecapabilities of assessing the tumor-specific immune response which, likeinfectious disease responses, consists of multiple effectors includingCD8⁺ T cells, CD4⁺ T cells, and antibodies. Knowledge of the extent andprevalence of tumor antigen-specific immunity can be used to identifywhich antigens are naturally targeted by the immune system and tounderstand why natural immunity fails to eradicate tumors. Severalmechanisms are proposed to explain immune escape.

The results provided herein also demonstrate that patients can generateimmunity to multiple epitopes suggesting that tolerance to FRα is absent(i.e., immunologic ignorance) or reversible (i.e., anergy). The resultsdemonstrate the existence of multi-epitope FRα-specific immuneresponses, suggesting that the T cell receptor repertoire targeting FRαis largely intact and is maintained in normal healthy individuals byeither ignorance or anergy. This is likely due to the fact that inhumans expression of FRα is limited to a few tissues, mainly kidneytubules. The observation that patients with breast and ovarian cancerapparently augmented immunity to the FRα, particularly to epitopes inthe amino terminal half of the molecule, shows that the immune systemmaintains a diverse T cell repertoire that can be expanded in vivo.

Although the polypeptides used herein were predicted CD4⁺ T cellepitopes, results suggest that some responses were due to CD8⁺ T cellsthat were potentially activated by encompassed WIC class I polypeptidessuch as with polypeptide FR56. In addition, one of the fourteenpolypeptides fully contained an HLA-A2-restricted epitope. Thatpolypeptide, FR238, fully encompasses the HLA-A2 motif, FRα 245-253. Inthe current study, 23% of patients were found to respond to FR238 whilenone of the healthy donors responded. Thus, although HLA-A2 expressionwas not examined, the possibility exists that the patients wereresponding to the embedded HLA-A2-restricted epitope.

The analyses provided herein suggest that the polypeptides may encompassepitopes that bind to other WIC class I molecules as well (e.g. HLA-A3).Polypeptides that could generate both CD4⁺ and CD8⁺ T cells are usefulfor generating an effective anti-tumor immune response since severalstudies have shown that activating both T cell subsets may be betterthan activating either alone. Coupled with detection of FRα-specificantibodies, the presence of both CD4⁺ and CD8⁺ T cell immunity indicatesthat a coordinated immune response is being elicited in cancer patients,but that the response may be limited.

The FRα is a tumor-associated antigen that may have a role in thebiology of cancer which may explain why it is maintained in a highproportion of tumors. For example, decreasing FRα expression in breastcancer cell lines reduces their proliferation rate. In addition, itshigh frequency of expression in ovarian cancer (>90%) suggests that FRαconfers a growth advantage over tumor cells with reduced expression.Coupled with the observations that the T cell repertoire is intact,these findings suggest that targeting the FRα using immune-basedapproaches such as cancer vaccines may be advantageous because theimmune system would target the most aggressive tumor cells. Targetingantigens that are involved in the biology of the disease may reduce therisks of outgrowth of antigen-negative variants.

In summary, the results provided herein demonstrate that immunity to FRαis prevalent in patients with breast and ovarian cancer. Understandingimmunity to tumor-associated antigens should lead to a betterunderstanding of how tumors interact and escape natural immunity.Furthermore, discovery of the epitopes of a tumor antigen such as theFRα could lead to design and testing of strategies to augmenttumor-specific immunity.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for increasing FRα immunity in a mammal,said method comprising administering a composition to said mammal underconditions effective to increase said FRα immunity, wherein saidcomposition comprises FR30.
 2. The method of claim 1, wherein saidmammal is a human.
 3. The method of claim 1, wherein said compositioncomprises an adjuvant.
 4. The method of claim 1, wherein saidcomposition comprises 500 μg of said FR30.
 5. The method of claim 1,wherein said FR30 comprises a lipid soluble alkyl.
 6. A method forincreasing FRα immunity in a mammal, said method comprisingadministering a composition to said mammal under conditions effective toincrease said FRα immunity, wherein said composition comprises FR56. 7.The method of claim 6, wherein said mammal is a human.
 8. The method ofclaim 6, wherein said composition comprises an adjuvant.
 9. The methodof claim 6, wherein said composition comprises 500 μg of said FR56. 10.The method of claim 6, wherein said FR56 comprises a lipid solublealkyl.
 11. A method for increasing FRα immunity in a mammal, said methodcomprising administering a composition to said mammal under conditionseffective to increase said FRα immunity, wherein said compositioncomprises FR76.
 12. The method of claim 11, wherein said mammal is ahuman.
 13. The method of claim 11, wherein said composition comprises anadjuvant.
 14. The method of claim 11, wherein said composition comprises500 μg of said FR76.
 15. The method of claim 11, wherein said FR76comprises a lipid soluble alkyl.